The incidence of many debilitating conditions, disorders and diseases, especially Alzheimer's disease, Parkinson's disease, cardiac infarction, amyotrophic lateral sclerosis (ALS), organ transplantations, and stroke, is continuously increasing in ageing societies, and thus represents not only a major health problem but also a growing socio-economic burden. Yet, and in particular, treatment strategies to combat these diseases are inadequate or fail to exist entirely. One major underlying factor of many such conditions, disorders and diseases is the role of non-apoptotic regulated cell death, and the associated diversity of aberrant cellular processes, which ultimately lead to cellular demise.
Cell death has been traditionally classified as apoptosis or necrosis. While apoptosis is now known and used as a term to describe a small set of lethal signalling pathways, the mechanisms for which have been extensively studied, necrosis was, until relatively recently, considered an unregulated process of mere accidental cell death. Little effort had been made to study necrosis due to its believed unregulated nature. More recently, support for forms of regulated cell-death mechanism other than apoptosis have since been found, described and standardised nomenclature recommended (Galluzzi et al. (2012); Cell Death Diff. 19:107-20, especially Table 1 thereof), including those termed “regulated necrosis” and “necroptosis”; a specific regulated cellular necrosis mechanisms, discrete from apoptosis as described by Hitomi et al. (Cell 135:1311-23 (2008)) and Degterev and co-workers (Nat. Chem. Biol. 1:112-9 (2005)). Other forms of regulated cell death are described in Galluzzi et al. (2012), including certain tentative new names for very specific signalling pathways that lead to cell death such as “parthanatos”, “paraptosis” and several others (see references in Galluzzi et al. 2012). Another form of non-apoptotic regulated cell death includes “ferroptosis”, a non-apoptotic, iron-dependent, oxidative form of cell-death recently described by Dixon and co-workers (Cell 149:1060-72 (2012)). While necroptosis and ferroptosis share many features, differences between their phenotypes can be observed, and it is to be expected that additional regulated cell death modalities and lethal signalling pathways exist and may be described and defined separately to necroptosis etc.
However, evidence is mounting that oxidative stress, a state associated with a high level of reactive oxygen species (ROS), is a common denominator of many such non-apoptotic regulated cell-death processes (and also a specific form of apoptosis known as “caspase-independent apoptosis”, a pathway of regulated cell-death that operates in parallel to caspase-dependent apoptosis in response to multiple intracellular stress conditions), and in particular most neuronal dysfunction, ultimately resulting in neurodegeneration (Lin, M. T. & Beal, M. F., Nature 443, 787-795 (2006)). Oxidative stress, the imbalance between the generation and clearance of ROS, is a potent inducer of cell death. Increased levels of ROS, impaired ROS regulating systems and oxidatively modified proteins, lipids and DNA are all hallmarks of postmortem brain tissues from Alzheimer's, Parkinson's and ALS patients. ROS are also a major causative factor in the degeneration of neurons in stroke patients. Stroke is one prominent example of tissue damage caused by ROS following ischemia-reperfusion injury. Tissue damage due to ischemia-reperfusion injury is, however, not restricted to the central nervous system, it is also a hallmark of infarction (cardiac infarction being the most prevalent type of infarction) and an important complication in surgery with special emphasis on solid organ transplantation. Oxidative stress and/or non-apoptotic regulated cell death is associated with many other conditions, disorders and diseases, or is a symptom the result of or arising from such a condition, disorder and disease. Of particular importance is cell, tissue, organ or organism intoxication, such as circumstances which are the result of, arise from or are associated with drug treatment (e.g., kidney toxicity from cisplatin), drug overdose (e.g., liver toxicity from paracetamol), acute poisoning (e.g., from alcohol, paraquat or environmental toxins) or exposure to ionizing radiation. Other conditions, disorders and diseases result in a state that is associated with oxidative stress and/or non-apoptotic regulated cell death, and include head trauma, asphyxia, cold or mechanical injury and burns. Oxidative stress and/or non-apoptotic regulated cell death may also be related to aesthetic conditions such as UV-damage/aging in skin and hair loss.
Oxidative stress-dependent cell death occurs frequently in a regulated fashion. Although there is no generalized consensus on the use of the expression ‘necroptosis’ (Vandenabeele et al. (2010) Nat. Rev. Mol. Cell Biol. 11:700-14), the terms ‘regulated necrosis’ and ‘necroptosis’ and ‘ferroptosis’ (Dixon et al., Cell, 2012) are used herein and known in the art to indicate general and specific forms (respectively) of regulated—as opposed to accidental—necrosis (Galluzzi et al. 2012). As indicated above, for a long time, necrosis was considered merely as an accidental uncontrolled form of cell death, but evidence that the execution of some forms of necrotic cell death is also finely regulated by a set of signal transduction pathways and catabolic mechanisms is further accumulating (Galluzzi and Kroemer, Cell 135 26:1161-1163 (2008); Kroemer et al., Cell Death Differ.; 16(1): 3-11, (2009)). For instance, death domain receptors (e.g., TNFR1, Fas/CD95 and TRAIL-R) and Toll-like receptors (e.g., TLR3 and TLR4) have been shown to elicit necrotic cell death, in particular in the presence of caspase inhibitors—strong evidence of the non-apoptotic nature of regulated necrosis and necroptosis. TNFR1-, Fas/CD95-, TRAILR- and TLR3-mediated cell death seemingly depends on the kinase RIP1, as this has been demonstrated by its knockout/knockdown and chemical inhibition with necrostatin-1. While little is currently known about the molecular mechanism of ferroptosis, this form of non-apoptotic regulated cell-death is characterised by the overwhelming, iron-dependent accumulation of lethal lipid ROS, and in at least some cells, NOX family enzymes make important contributions to this process, and Dixon et al. postulate that the executioners of death in certain cancer cells undergoing ferroptosis are these ROS themselves.
Although there is no consensus on the biochemical changes that may be used to unequivocally identify oxidative stress-dependent or non-apoptotic regulated cell-death, several mediators, organelles and cellular processes have already been implicated in such cell death (Kroemer et al., Cell Death Differ.; 16(1): 3-11, (2009)). These phenomena include mitochondrial alterations (e.g., uncoupling, production of reactive oxygen species, i.e., ROS, nitrosative stress by nitric oxide or similar compounds and mitochondrial membrane permeabilization, i.e., MMP, often controlled by cyclophilin D), lysosomal changes (ROS production by Fenton reactions, lysosomal membrane permeabilization), nuclear changes (hyperactivation of PARP-1 and concomitant hydrolysis of NAD+), lipid degradation (following the activation of phospholipases, lipoxygenases and sphingomyelinases), increases in the cytosolic concentration of calcium (Ca2+) that result in mitochondrial overload and activation of noncaspase proteases (e.g., calpains and cathepsins). It is still unclear, though, how they interrelate with each other.
Notwithstanding, a crucial role for the RIP (receptor interacting protein) kinases, in particular serine/threonine kinases RIP1 and RIP3, has been demonstrated for regulated necrotic cell death (Declerq et al., Cell 138:229-232 (2009)). The multiprotein complex comprising RIP1 and RIP3 is known in the art as “necrosome”. RIP1 and RIP3 form the core complex within the necrosome. The necrosome complex further comprises TRADD and FAS-associated protein with a death domain (FADD), caspase 8, the serine/threonine-protein phosphatase (PGAM5) (Micheau et al., Cell 14:1814-190 (2003) and Wang et al. (2012), Cell 148:228-243) and the mixed lineage kinase domain-like protein (MLKL) (Sun et al. (2012), Cell 148:213-227). The necrosome regulates the decision between cell survival and regulated necrosis. In more detail, the phosphorylation of RIP1 and RIP3 engages the effector mechanism of regulated necrosis. In contrast, if caspase 8 is activated, it cleaves RIP1 and RIP3 thereby preventing the effector mechanism of regulated necrosis (Vandenabeele et al., Nature Reviews Mol. Cell Biol., 11:700-714 (2010)). Accordingly, the activation status of caspase 8 appears to be decisive whether a cell undergoes regulated necrosis or apoptosis by the initiation of the pro-apoptotic caspase activation cascade. Whether FADD or TRADD are strictly required for the assembly of the necrosome is presently not clear.
Besides caspase 8, negative regulators of TNR-receptor-family- or Toll like receptor-mediated regulated necrosis include E3 ubiquitin ligases cIAP1 and cIAP2, cFLIP, and TAK1, whereas the deubiquitinating enzymes CYLD and A20 act as positive regulators of regulated necrosis (Vandenlakker et al, Cell Death Differ 18, 656-665, 2011). Remarkably, the long and short isoforms of cFLIP were shown to act antagonistically, the short isoform promoting and the long isoform inhibiting TLR-ligand induced regulated necrosis (Feoktistova et al., Mol. Cell 43, 449-463, 2011). FAB2 and FAB2 are additional components of the signalling complex formed upon TNF receptor-ligation whose precise function in the regulation of regulated necrosis is still unknown. TRIF, an adapter protein with a RIP1 homology interaction motif (RHIM) is coupling the signalling complex formed upon Toll like receptor ligation to TLR3 and TLR4.
A RIP1 and RIP3 containing multiprotein complex promoting apoptosis or regulated necrosis is formed independently from TNF receptor family members in response to DNA damage-mediated depletion of cIAP1 and cIAP2. This complex further comprises FADD and caspase 8, the latter being the decisive determinant for the choice between apoptosis and regulated necrosis (Tenev et al., Mol. Cell 43, 432-448, 2011).
RIP1 is found in several types of complexes mediating an innate immune response to RNA and DNA viruses. A complex comprising TANK, FADD, TRADD, NEMO, and RIP1 is recruited to the outer membrane of mitochondria in response to ligation of pattern recognition receptors RIG-I or MDA5 recognizing viral RNAs through interaction with IPS1 (also called MAVS). RIP1 shares RIP1 homotypic interaction motifs (RHIM) for dimerization not only with RIP3, but also with the cytosolic DNA sensor DAI and TRIF (the latter being involved in signal transduction through TLR3 and TLR4). As exemplified for the murine cytomegalovirus protein M45, proteins or peptides containing a RHIM sequence may disrupt the RHIM interaction between RIP1 and RIP3 and may thus inhibit regulated necrosis (consensus sequence: I/V-Q-I/L/V-G-x-x-N-x-M/L/I)(Mack et al., PNAS 105, 3094-3099, 2008; Kaiser et al., J. Immunol. 181, 6427-6434, 2008). Although regulated necrosis promoting activities have not been reported so far for the sensors of viral RNA and DNA, these RIP1 containing protein complexes might nevertheless operate as molecular switches for oxidative signals that convert a pro-survival (or an interferon-inducing) signal into a regulated necrosis inducing signal.
In respect of ferroptosis, Dixon and co-workers (2012) cannot exclude the possibility of a death-inducing protein or protein complex that is activated downstream of ROS accumulation as observed for that form of non-apoptotic regulated cell-death.
Accordingly, regulated necrosis (and potentially other related forms of non-apoptotic regulated cell-death such as ferroptosis) may be characterized as a type of cell death that can be avoided by inhibiting—either directly or indirectly—the necrosome or other components of ferroptotic signalling, in particular the activity and/or interaction of components thereof such as RIP1, RIP3 and others such as one or more members of the ferroptotic pathway (either through genetic or pharmacological methods). This represents a convenient means to discriminate between regulated necrosis (e.g., necroptosis) and accidental forms of necrosis (Kromer et al., Cell Death Differ.; 16(1): 3-11, (2009)).
Certain spiroquinoxaline derivatives, and pharmaceutical compositions thereof, are generically disclosed in EP 0 509 398A1 and EP 0 657 166 A1, in particular for use in the treatment of virus infection.
Together with a very large number of other generic structures, certain spiroquinoxaline derivatives are generically disclosed in WO 2007/117180 A1 as formula 2.2, and a small number of specific spiroquinoxaline compounds are disclosed in the expansive Table 12 thereof. WO 2007/117180 A1 relates primarily to the construction and composition of large combinatorial libraries of small molecules having interest as merely potential physiologically active substances and pharmaceutical compositions thereof. Of the huge number of specific compounds disclosed therein, only a small number of compounds (not being spiroquinoxalines) are tested for and suggested to have anti-cancer properties (example 40 and Table 14 thereof).
Frankowski and co-workers (PNAS 108:6727-32 (2012)) describe the synthesis of a small library of Stemona alkaloid analogues (reported as having antitussive activity) that are fused by a spiro-carbon to quinoxaline derivatives (Scheme 4 therein), and the activity of such alkaloid-quinoxalines spiro-fusions in various receptor-binding assays.
Various organic chemistry methods are disclosed for the synthesis of quinoxaline derivatives and spiro forms thereof. These include, Kysil et al (Eur. J. Org. Chem. 8:1525-43 (2010)), Lee (KR 2012-105714), Adarvana (Tetrahedron Lett. 52:6108-12 (2011)), and Kolla and Lee (Tetrahedron 66:8938-44 (2010)).
Other specific spiroquinoxaline compounds are known and are commercially available, but without indication of synthesis or utility. These include those with CAS registry numbers: 1172351-24-4, 1223830-23-6 and 1223982-82-8.
Accordingly, it is an object of the present invention to provide alternative, improved and/or integrated means or methods that address one or more problems, including those described above such as in the treatment (including prophylactic treatment) of one or more conditions, disorders or diseases (or related conditions or symptoms) and/or agents and pharmaceutical compositions useful for such treatment. Such an object underlying the present invention is solved by the subject matter as disclosed or defined anywhere herein, for example by the subject matter of the attached claims.